The primary objective of this project is to develop, using a novel vaccine platform technology (the VesiVax(r) system), a safe HSV2 vaccine that is capable of providing a level of protection in male and female human populations which is better than other recombinant vaccines. We have shown that HSV2 VesiVax(r) vaccines targeted to single epitopes from the gD envelope protein are capable of protecting both female and male mice from mucosal HSV2 challenge (intravaginal and intrarectal, respectively) with increased survival and markedly reduced clinical signs of infection and further, actively stimulates effective T-cell immune responses in both sexes. In the Phase I studies, we demonstrated that an antigenic segment corresponding to the entire HSV2 gD ectodomain (amino acids 1-306) provided protection from disease in both intravaginal (female) and intrarectal (male and female) mouse models of lethal HSV2 challenge with a significant reduction in primary disease and a trend towards reducing recurrent infections in a guinea pig model. We now propose to take our lead vaccine candidate, L-HSV2gD1-306-HD, and move it towards clinical evaluation. To accomplish this objective, a number of technical objectives will need to be completed. Our first step will be to determine the optimal antigen dose and schedule as well as different routes of administration (i.e., subcutaneous or intramuscular) required for effective immunization in male and female mice challenged intravaginally or intrarectally with a lethal dose of HSV2. The L-HSV2gD1-306-HD vaccine will undergo further testing by immunizing male and female guinea pigs with increasing antigen doses at the optimal dosing schedule and route of administration determined from the mouse studies and then challenging the animals with HSV2 to determine their response to primary and recurrent infection. We will also characterize the immunological response of the male and female mice to the L-HSV2gD1-306-HD vaccine construct using the optimized vaccination parameters. Concurrently, with the optimization studies, we will focus on developing the processes for manufacturing clinical grade batches of the L-HSV2gD1-306-HD vaccine. Our first activities will be directed towards producing a Development Cell Bank. We will then prepare a Master Cell Bank and Working Cell Bank from the Development Cell Bank and characterize them. We will then optimize the HSV2gD1-306-HD production process by fermentation using standard equipment that is amenable to scale-up. The processes for isolating and purifying the HSV2gD1-306-HD protein will be based on modifications of existing methods and the development of the processing conditions to produce the L-HSV2gD1-306-HD vaccine in larger scale will be performed. We will also develop the analytical methods required for ensuring the quality of each preparation of L-HSV2gD1-306-HD. Using these assays, batches of HSV2gD1-306-HD will be evaluated for stress testing. Using these assays, batches of L-HSV2gD1-306-HD will be monitored under different conditions (i.e., temperature, pH) for stress testing. Using these procedures, the L-HSV2gD1-306-HD vaccine will be scaled-up and pilot batches of material produced in conformance to FDA current Good Manufacturing Practice (cGMP) regulations. The pilot batches of L-HSV2gD1-306-HD will be prepared in sufficient amounts to allow for stability and equivalency testing, and eventual safety and immunogenicity studies. For the stability studies, vials of L- HSV2gD1-306-HD will be stored at selected storage temperatures and analyzed periodically for changes in the product. Lastly, the pilot batches of material will be tested in the male and female mouse models of HSV2 infection that have been used to develop the L-HSV2gD1-306-HD vaccine. The pilot material will be compared against laboratory prepared material in terms of survival and reduction of the signs of disease following challenge with a lethal dose of HSV2. The pilot batches of material will also be tested in the male and female guinea pig models of recurrent HSV2 infection that have been used to develop the L-HSV2gD1-306-HD vaccine. The pilot material will be compared against laboratory prepared material in terms of reduction of the primary infection following challenge with an infectious dose of HSV2 and the lack of recurrence of herpetic lesions. PUBLIC HEALTH RELEVANCE: This SBIR research will lead to the development and production of a novel genital herpes vaccine having significant advantages in production economy and immunological activity for future clinical use in preventing the spread of HSV2. [unreadable] [unreadable] [unreadable]